Effect of bacterial and cyanobacterial culture on growth, quality and ...

Proceedings <strong>of</strong> the 7 th International Conference on Mushroom Biology <strong>and</strong> Mushroom Products (ICMBMP7) 2011
EFFECT OF BACTERIAL AND CYANOBACTERIAL CULTURE ON
GROWTH, QUALITY AND YIELD OF Agaricus bisporus
HOSSEIN RIAHI, A. ESKASH, Z. SHARIATMADARI
Faculty <strong>of</strong> Biosciences, Shahid Beheshti University G, C.
Evin,Tehran,
Iran
H-Riahi@cc.sbu.ac.ir
ABSTRACT
In the mushroom growing process, Agaricus bisporus function <strong>of</strong> casing soil is to provide an
environment for fruit formation. Presence <strong>of</strong> Pseudomonas species in casing soil is important for
mushrooms formation <strong>and</strong> development. They can represent up to 50 percent <strong>of</strong> the total
bacteria. The result <strong>of</strong> this study showed that total number <strong>of</strong> bacteria <strong>and</strong> population <strong>of</strong>
pseudomonas species increased dramatically after casing. The maximum number <strong>of</strong> bacteria was
recorded at the primordia formation stage. Population <strong>of</strong> pseudomonas in casing soil was
increased with addition <strong>of</strong> <strong>bacterial</strong> inoculum to the casing soil. There was a significant
difference <strong>of</strong> mushroom yield as compared to the control.
Until now most research <strong>and</strong> applications <strong>of</strong> cyanobacteria have been conducted with green
plants growing. Findings <strong>of</strong> this research strongly supported that the production <strong>of</strong> promoting
substances such as auxins, sugars <strong>and</strong> vitamins by the algae may be partly responsible for the
greater mushroom growth <strong>and</strong> yield. Inoculation <strong>of</strong> cyanobacteria into the casing soil
significantly increased mushroom yield <strong>and</strong> quality.
Keywords: Agaricus; Pseudomonas; Primordial; Cyanobacteria; Phytohormones
INTRODUCTION
Following colonization <strong>of</strong> mushroom mycelia in pasteurized compost, a 1.5 inch layer called
casing soil is applied on top <strong>of</strong> the compost surface. The casing soil enhances the retention <strong>of</strong>
irrigation water on production beds <strong>and</strong> promotes mushroom fruit body formation. An important
process during mushroom growing is pinning. Addition <strong>of</strong> casing is necessary for shifting the
vegetative phase to the reproductive phase by pin formation. The casing soil supports an active,
aerobic <strong>bacterial</strong> flora [2] among them fluorescent Pseudomonas spp. play an important role in
initiation <strong>of</strong> pinning <strong>and</strong> mushroom fruit body development. There is a controversy regarding
populations <strong>of</strong> pseudomonads in casing soil. Samson [12] demonstrated that fluorescent
pseudomonads may represent up to 50 percent <strong>of</strong> the total bacteria in casing samples, whereas
Doores et al. [1] indicated that they represented only 2 percent <strong>of</strong> the total casing bacteria. Miller
et al. [7] showed that populations <strong>of</strong> casing bacteria changed over the A. bisporus growth cycle.
Pseudomonas putida has been identified as an important species involved in fruiting body
initiation [3, 10]. Inoculation <strong>of</strong> <strong>bacterial</strong> <strong>culture</strong> on mushroom growing media, besides the effect
on pin formation, caused an increase <strong>of</strong> mycelial growth rate up to 1.6 fold, promoted the rate <strong>of</strong>
radial hyphal extension, <strong>and</strong> suppressed the frequency <strong>of</strong> branching [6].
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Proceedings <strong>of</strong> the 7 th International Conference on Mushroom Biology <strong>and</strong> Mushroom Products (ICMBMP7) 2011
Soil is a habitat <strong>of</strong> some terrestrial blue-green algal species that are beneficial organisms for
soil fertility by fixing atmospheric nitrogen, binding soil particles, helping to maintain moisture
<strong>and</strong> preventing erosion. These bacteria supply substances that promote the growth <strong>of</strong> plants.
There are many reports that cyanobacteria produce phytohormones such as cytokinin, auxin <strong>and</strong>
auxin-like substances in soil [5, 13, 14, 15, 16]. Other plant growth regulator (PGR) substances
such as amino acids, sugars, vitamins that may have a positive influence on growth <strong>of</strong> vascular
plant are produced by cyanobacteria [5, 8, 9]. Until now most research <strong>and</strong> applications <strong>of</strong>
cyanobacteria have been conducted with green plants. This is a first attempt to study the effect <strong>of</strong>
cyanobacteria on mushroom. Moreover, cyanobacteria may enhance production <strong>of</strong> secondary
metabolites. These phenomena may be controlled with or mediated by hormones [11, 13].
Cyanobacteria are another group <strong>of</strong> microorganisms that might have a positive effect on
mushroom yield.
MATERIALS AND METHODS
In this work two experiments were conducted. The first study concerned the <strong>bacterial</strong> population
in casing soil <strong>and</strong> effects <strong>of</strong> Pseudomonas patuda used as <strong>bacterial</strong> inoculum, on yield <strong>and</strong>
quality <strong>of</strong> mushroom. A similar objective was followed in the second trial but algal <strong>culture</strong> was
used for inoculation.
Experiment 1. Isolation <strong>and</strong> cultivation <strong>of</strong> Pseudomonas species from casing soil. After
colonization <strong>of</strong> compost with mushroom mycelium, the substrate was covered with casing soil.
Sampling was carried out in Malard mushroom research farm. To determine Pseudomonas
population, casing soil was sampled periodically until primordia formation. Specified amount <strong>of</strong>
the dilutions transferred on to sterile plates <strong>of</strong> nutrient agar (NA) <strong>and</strong> Chromagar Pseudomonas
(PS822) media. Inoculated plates were incubated at 30 o C for 24h. Colonies that appeared at the
end <strong>of</strong> incubation were counted, the unit expressed in terms <strong>of</strong> colony-forming units per gram
(CFU/g) <strong>of</strong> original sample. The isolates were further subjected to st<strong>and</strong>ard biochemical tests.
Bacterial identification <strong>of</strong> isolates was carried out by comparing the results obtained with
Bergey’s Manual <strong>of</strong> Determinative Systematic Bacteriology. The predominant Pseudomonas
species isolated from casing soil was tested to study the growth <strong>of</strong> mycelium <strong>and</strong> pin formation.
Inocula were prepared by growing the selective strains in nutrient broth (NB) medium.
After incubation at 30 o C for 18h, the densities <strong>of</strong> <strong>culture</strong> were determined at OD600. Then
<strong>culture</strong>s were diluted further in serum until final <strong>bacterial</strong> cell numbers were 10 8 cells/ml.
Bacterial suspensions (5 L) were sprayed on 42 m 2 <strong>of</strong> casing soil at time <strong>of</strong> casing.
Protein contents <strong>of</strong> fruit bodies were calculated from the nitrogen content (N×6.25) as
determined by micro-Kjeldahl method [4].
Experiment 2. Cyanobacteria cultivation. Among the cyanobacteria isolated from different
paddy fields one heterocyestous cyanobacteria (Nostoc) was selected because it showed a high
growth rate. Isolates were grown in BG11 nitrogen free medium in a 2 L container at 24°C <strong>and</strong> a
12/12 h light/dark cycle with artificial illumination (2000-2500 Luxes) <strong>and</strong> constant stirring <strong>and</strong>
aeration. After three weeks, the <strong>culture</strong> was harvested <strong>and</strong> used as inoculum. Addition <strong>of</strong> algal
<strong>culture</strong> was carried out at the primordia formation stage, before second <strong>and</strong> third flushes.
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Proceedings <strong>of</strong> the 7 th International Conference on Mushroom Biology <strong>and</strong> Mushroom Products (ICMBMP7) 2011
Mushroom production. The compost formulation consisted <strong>of</strong> wheat straw, chicken manure
<strong>and</strong> gypsum prepared in conventional yard <strong>and</strong> pasteurization tunnels. Mushrooms were grown
in controlled <strong>and</strong> st<strong>and</strong>ard growing room. After colonization <strong>of</strong> compost with A. bisporus
(commercial strain Sylvan A15) mycelium, the substrate was covered with 4-5 cm <strong>of</strong> casing soil.
Mushroom yield was determined over a 3-weeks production period. Mushrooms were weighted
after removal <strong>of</strong> stipes.
Statistical analysis. Data were subjected to analysis <strong>of</strong> variance (ANOVA) <strong>and</strong> means were
compared using LSD test (P

Proceedings <strong>of</strong> the 7 th International Conference on Mushroom Biology <strong>and</strong> Mushroom Products (ICMBMP7) 2011
percent in the third break. Protein content <strong>of</strong> treated mushrooms also increased with 3 to 5
percent in three breaks compared to untreated mushrooms (Table 1).
Table 1. <strong>Effect</strong> <strong>of</strong> <strong>bacterial</strong> <strong>culture</strong> on yield, dry matter <strong>and</strong> protein content <strong>of</strong> mushroom
First picking Second picking Third picking
Sample
Yield D.M Protein
Kg/m 2 % %
Yield D.M Protein
Kg/m 2 % %
Yield D.M Protein
Kg/m 2 % %
Control 11.8 8.2 37 7.9 7.9 34 4.8 7.5 32
Test 12.1 ns 8.7 * 40* 10 ** 8.5 * * 4.4 ns 8.2 * *
39 **
37 **
Diff. 0.3 0.5 3.0 2.1 0.6 5.0 - 0.4 0.7 5.0
* * s i g n i f i c a n c e < 1 % l e v e l , * s i g n i f i c a n c e < 5 % l e v e l , n s :
n o t s i g n i f i c a n t
Casing soil was irrigated with algal <strong>culture</strong> at the primordia formation stage, before
second <strong>and</strong> third breaks. There was a significant difference in mushroom yield in three flushes
treated with cyano<strong>bacterial</strong> <strong>culture</strong> as compared to the control. Mushroom yield was increased
0.661 kg/m 2 in first, 2.2 kg/m 2 in second flush <strong>and</strong> 0.053 kg/m 2 in the third flush as compared to
the control. The result also showed that inoculation <strong>of</strong> casing soil with cyanobacteria had a
positive effect on quality <strong>of</strong> mushroom. Dry matter content <strong>of</strong> mushrooms increased in the first
flush. Dry matter was measured at 9.75 % in treated <strong>and</strong> 8.35 % in untreated mushroom.
However, the amount <strong>of</strong> dry matter <strong>and</strong> protein declined in second <strong>and</strong> third flush as compared to
the first flush. There was a slight difference in dry matter in treated <strong>and</strong> untreated mushroom in
second <strong>and</strong> third flushes. Addition <strong>of</strong> algal <strong>culture</strong> had a positive effect on protein content <strong>of</strong>
mushroom. The amount protein was increased significantly in first flush, but there was a slight
difference between treated <strong>and</strong> control in second flush. Whereas, in the third flush protein
content <strong>of</strong> treated mushroom was less than control (Table 2).
Table 2. <strong>Effect</strong> <strong>of</strong> algal <strong>culture</strong> on yield, dry matter <strong>and</strong> protein content <strong>of</strong> mushroom
Sample First Flush Second Flush Third Flush
Yield D.M Protein
Kg/m 2 % %
Yield D.M Protein
Kg/m 2 % %
Yield D.M Protein
Kg/m 2 % %
Control 12.59 8.35 48.1 7.58 8.3 45.1 2.75 7.40 41.1
Test 13.2 * 9.75 * *
51.9 **
9.78 ** 8.6 *
45.5 ns
2.80 ns 7.50 ns
39.1 ns
Diff. 0.61 1.4 3.8 2.2 0.3 0.4 0.05 0.10 -2.0
* * s i g n i f i c a n c e < 1 % l e v e l , * s i g n i f i c a n c e < 5 % l e v e l ,
n s : n o t s i g n i f i c a n t
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CONCLUSION
In cultivation <strong>of</strong> A. bisporus, compost <strong>and</strong> casing soil are two major elements. Studies
have demonstrated that populations <strong>of</strong> pseudomonas in the casing layer on which the mushroom
fruit body develops is very important. In most <strong>of</strong> the studies, total <strong>bacterial</strong> populations ranged
from 8.0 to 8.5 log CFU/g casing material. With a slight difference, this result is same as other
workers. The majority <strong>of</strong> <strong>bacterial</strong> population in casing was attributed to pseudomonas species.
In this study they present more than 80 present <strong>of</strong> the <strong>bacterial</strong> population in the casing layer.
The result showed that there is a close relation between growth <strong>of</strong> mycelium <strong>and</strong> number <strong>of</strong>
bacteria in casing soil. Sampling <strong>of</strong> casing soil at different periods <strong>of</strong> mushroom growing cycle
revealed that number <strong>of</strong> bacteria increased simultaneously with increase growth <strong>of</strong> mycelium
into casing soil. At the pining stage, populations <strong>of</strong> pseudomonas species especially P. putida
were important since they are playing a key role in fruiting body formation. We conclude that
inoculation <strong>of</strong> native P. putida isolated from casing soil at the primordia formation stage will be
very efficient for increasing mushroom yield <strong>and</strong> quality.
In the second experiment the application <strong>of</strong> cyano<strong>bacterial</strong> <strong>culture</strong> on casing soil <strong>and</strong> its effect on
mushroom yield <strong>and</strong> quality was investigated. Irrigation <strong>of</strong> casing soil with cyano<strong>bacterial</strong>
<strong>culture</strong> increased yield, dry matter <strong>and</strong> protein content <strong>of</strong> mushrooms. Production <strong>of</strong> promoting
substances such as auxins, sugars <strong>and</strong> vitamins by the algae are the main factors for this issue.
ACKNOWLEDGEMENTS
We are very much thankful to the managers <strong>and</strong> staff <strong>of</strong> Malard mushroom company –
especially Mr. Kabei, Judy, Soltan Shah, Marzoughi <strong>and</strong> Ebrahimi for their assessment. Without
their help we would not be able to carry out this research. The authors are also grateful to the
University <strong>of</strong> Shahid Beheshti for financial support.
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